The present invention relates to a process for extracting conjugated estrogens from pregnant mare urine. The present invention further relates to a process for obtaining a natural mixture of conjugated estrogens. The mixture of conjugated estrogens can be used to prepare products for estrogen replacement therapy or hormone replacement therapy.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text and respectively grouped in the appended Bibliography.
Menopause is generally defined as the last natural menstrual period and is characterized by the cessation of ovarian function, leading to the substantial diminution of circulating estrogen in the bloodstream. Menopause is usually identified, in retrospect, after 12 months of amenorrhea. It is not a sudden event, but is often preceded by a time of irregular menstrual cycles prior to eventual cessation of menses. Following the cessation of menstruation the decline in endogenous estrogen concentrations is typically rapid. There is a decrease in serum estrogens from circulating levels ranging from 40-250 pg/mL of estradiol and 40-170 pg/ml of estrone during ovulatory cycles to less than 15 pg/mL of estradiol and 30 pg/mL of estrone in postmenopausal women.
As these estrogens decline during the time preceding (perimenopuase) and following the menopause (postmenopause), various physiological changes may result, including vulvar and vaginal atrophy causing vaginal dryness, pruritus and dysparenuia, and vasomotor instability manifested as hot flushes. Other menopausal disturbance may include depression, insomnia, and nervousness. The long-term physiologic effects of postmenopausal estrogen deprivation may result in significant morbidity and mortality due to increase in the risk factors for cardiovascular disease and osteoporosis. Menopausal changes in blood lipid levels, a major component of the pathogenesis of coronary heart disease (CHD), may be precursors to increased incidence of ischemic heart disease, atherosclerosis, and other cardiovascular disease. A rapid decrease in bone mass of both cortical (spine) and trabecular (hip) bone can be seen immediately after the menopause, with a total bone mass loss of 1% to 5% per year, continuing for 10 to 15 years.
Estrogen replacement therapy (ERT) is beneficial for symptomatic relief of hot flushes and genital atrophy and for prevention of postmenopausal osteoporosis. ERT has been recognized as an advantageous treatment for relief of vasomotor symptoms. There is no acceptable alternative to estrogen treatment for the atrophic changes in the vagina; estrogen therapy increases the vaginal mucosa and decreases vaginal dryness. Long term ERT is the key to preventing osteoporosis because it decreases bone loss, reduces spine and hip fracture, and prevents loss of height. In addition, ERT has been shown to be effective in increasing high density Lipoprotein-cholesterol (HDL-C) and in reducing low density lipoprotein cholesterol (LDL-C), affording possible protection against CHD. ERT also can provide antioxidant protection against free radical mediated disorders or disease states. Estrogens have also been reported to confer neuroprotection, and inhibit neurodegenerative disorders, such as Alzheimer's disease. The best known oral estrogen replacement therapy available in the United States is a natural mixture of conjugated equine estrogens sold under the trademark Premarin.
To minimize the occurrence of estrogen-related side effects and to maximize the benefit-risk ratio, the lowest dose effective in relief of symptoms and prevention of osteoporosis should be used. Although ERT reduces the relative risk for ischemia heart disease and osteoporosis, the relative risk of endometrial cancer for postmenopausal women with a uterus may be increased. There are extensive clinical data showing that the relative risk of endometrial cancer can be reduced by the addition of a progestin, either sequentially or continuously. The addition of a progestin to estrogen therapy prevents estrogen-induced endometrial proliferation. Continuous combined hormone replacement therapy (HRT), with appropriate dose of daily estrogen and progestin, has been shown to be effective in relieving vaginal atrophy and vasomotor symptoms, preventing postmenopausal osteoporosis, and reducing the risk of endometrial cancer by prevention of endometrial hyperplasia. See for example, U.S. Published Patent Application No. 20010034340.
As noted, natural mixtures of conjugated estrogens such as are found in the urine of pregnant mares have proved particularly effective and well tolerated for ERT and HRT. The dissolved solids content in the urine of pregnant mares (i.e., pregnant mare urine; PMU) may naturally vary within wide ranges, and may generally lie in a range of 40-90 g dry matter per liter. In addition to urea and other usual urine contents, phenolic constituents are contained in the solids content of the PMU in quantities of about 2%-5% by weight related to dry matter. These phenolic constituents include cresols and dihydro-3,4-bis[3-hydroxyphenyl)methyl]-2(3H)-furanone (HPMF). These phenolics may be present in free or conjugated form. The PMU contains a natural mixture of estrogens which is largely present in conjugated form, e.g. as sulfuric acid semi-ester sodium salt (referred to hereinafter as “sulfate salt”). The content of conjugated estrogens (calculated as estrogen sulfate salt) may be between 0.3% and 1% by weight relative to dry matter.
Usually extracts containing conjugated estrogens are obtained from the PMU by extraction with a polar organic solvent which is immiscible, or only slightly miscible, with water, such as ethyl acetate, n-butanol or cyclohexanol. In such liquid-liquid extractions, however, a number of problems occur, such as severe foaming, sedimentation, emulcification and poor phase separation. Generally several extraction steps are required, which results in losses and only partial recovery of the estrogen content.
Conjugated estrogens have been isolated by extracting PMU and an organic solvent such as n-butanol or instead by adsorption on charcoal. Such methods have involved a multiplicity of individual process operations, involving back extraction and repeated transfer between n-butanol and aqueous solutions. Such repeated extractions generally result in losses of conjugated estrogens and thus only partial recovery of the estrogen content of the PMU. Examples of an extraction process are discloses in U.S. Pat. Nos. 2,696,265; 2,711,988 and 2,834,712.
In 1968 it was proposed by Bradlow (1968) to use Amberlite XAD-2, a neutral, non-polar hydrophobic polystyrene resin, manufactured by Rohm and Haas, for extraction of conjugated estrogens from urine. The adsorption capacity given is low. According to Bradlow, an optionally diluted urine is passed through a column containing the resin at a low rate of flow. The estrogens are eluted with methanol or ethanol.
Other solid phase extractions of conjugated estrogens from PMU have been described in U.S. Pat. Nos. 3,769,401, 5,723,454 and 5,814,624. In U.S. Pat. No. 3,769,401, a polyamine anion exchange resin, such as Dowex 1-x-2, is used for extracting conjugated estrogens from PMU. The conjugated estrogens adsorbed on the column is eluted with methanol. U.S. Pat. No. 5,723,454 describes the use of a semi-polar polymeric adsorber resin to extract conjugated estrogens from filtered PMU. The adsorbed conjugated esters are desorbed with a water-miscible organic solvent, such as ethers, aliphatic alcohols or ketones. The resin is a porous organic non-ionic polymer, such as a cross-linked aliphatic polycarboxylic acid ester resin, such as Amberlite XAD-7 polyacrylic acid ester resin. U.S. Pat. No. 5,814,624 discloses the use of hydrophobized silica gel, also known as reverse-phase silica gel, to adsorb conjugated estrogens from PMU. The adsorbed conjugated esters are desorbed with a water-miscible organic solvent, such as ethers, alcohols or ketones.
Despite all the past activity in this field, there has remained a need in the art for a more effective way to recover estrogens from pregnant mare urine for preparing conjugated estrogen products.